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methyispiperone (13, 14), [‘8F]setoperone(15,16,17), [‘‘C]
methylbromo-LSD (18,19), and [‘8F]ritanserin(20,21).
Altanserin, like setoperone, is a fluorobenzoyl derivative
structurally related to ketanserin (22). The in vitro binding
affinity constants (Ki: nM) ofaltanserin for 52, D2, and a,
receptor sites, reported by J. E. Leysen (23) are respectively
0. 13, 62 and 4.55 (Table 1). Based on these findings,
altanserin was considered as potentially interesting radi
otracer for in vivo serotonin receptor binding.
In this paper, we reportthe radiosynthesisof [‘8F]altan
serin by nucleophilic fluorination of the corresponding
nitro derivative as precursor(Fig. 1). Rat experiments also
are described, including general and regional biodistribu
tions as well as carrier and blocking effects.
MATERIALS AND METHODS
StartingMaterials
Most of the organic substratesand solvents were of analytical grade from Aldrich. Methyl 2-isothiocyanatobenzoate, hydro bromic acid, hydriodicacid (57 %wt), and the gold label reagents dimethylsulfoxide and acetonitrile were purchased from Janssen
Chimica and used without further purification. The aminopo
lyether kryptofix 222 (4, 7,13,16,21,24) hexaoxa-l,lO diazabicy clo(8.8.8)hexacosan, potassium carbonate, and ethanol were oh tamed from Merck. Altanserin, bromoaltanserin, and nitroben
zoylpiperidinewerea gift from Janssen Pharmaceutica.Oxygen
18-enrichedwater (98.5%)was obtained from Campro Benelux.
All other reagents including @-chloroethyl-ethylcarbamate(24)
werepreparedaccordingto literature methods.
HPLCSystem
High-performance liquid chromatography (HPLC) was con ducted using a Waters system consisting of M-6000 A pump, a
U6K injectorand a Lambdamax 481 LC U.V. spectrophoto
meter set up at 254 nm with a 10-mmanalyticalcell.A Nal (Ti)
scintillation detector was used for radioactivity measurements.
LichrosorbRP Select-Bcolumns were from Merck. Conditions
(column,eluent, flow)for separationsare given in the following
sections.
Radiochemistry
The synthesis of [‘tF]altanserinwas performedas describedin the Appendix by nucleophilic substitution of the mtro group of
No-carrier-added[18Fjaltanserinwas synthesizedby nudeo philic substitution of the corresponding nitro compound with
[18F]fluorideinthe presence of kryptofix222 and K2C03.After
purificationby preparative HPLC, [18F]altansennwas pro duced in less than 2 hr with a radiochemical yield of 10% (EOS) and a specificactivityof 0.8—1.3 Ci/@mol.In rats, the tracer localized rapidly in the whole brain (0.5% 10/9 organ) with a highbindingto the frontalcortex. The frontalcortex/ cerebellum ratio increased with time and reached a plateau of 11 at 2 hr postinjection.This uptake in S2receptorregions was saturableand could be blocked by pretreatmentwith variousS2 antagonists.This radiopharmaceuticalappearsto be more selective for S2 receptor sites than other ligands available today and allows the study of S@receptors under in vivoconditions.
J Nucl Med 1991; 32:2266-
n vitro studies on human brain autopsy material have
shown that the serotonin receptor system is implicated in
several conditions, including sleep (1), aging (2), Alz
heimer's disease (3), affective and personality disorders
(4—6),pain (7), and alsoextrapyramidalsyndromes(8).
In vivo study of 52 receptorswould representa significant
advance in the understanding of the various conditions
where these sites are thought to play a role. Positron
emission tomography (PET) allows the direct, noninvasive
and repetitive measurement of neuroreceptors in regions
of the brain provided that the appropriate radioligand is
available at high specific activity (9).
A number of ‘‘C-or ‘8F-labeledradioligands have al
ready been synthesized as radiopharmaceuticals for map
ping 52 serotoninergic receptorsites with PET. To date, in
vivo studies have been performed with several serotoni
nergic antagonists labeled with positron emitters such as
[@C]ketanserin (10,11), [‘8F]spiperone(12) and [“C]
Received March 21 , 1991 ; revision accepted August 7, 1991. For reprints contact: Christian Lemaire, Cyclotron Research Center, Liege 1k@iversity,4000 Liege, Belgium.
(^2266) The Journalof NuclearMedicine•Vol. 32 •No. 12 •December
fluorine- 18-Altanserin: A Radioligand for the
Study of Serotonin Receptors with PET:
Radiolabeling and In Vivo Biologic Behavior
in Rats
Christian Lemaire, Robert Cantineau, Marcel Guillaume, Alain Plenevaux, and Leon Christiaens
Cyclotron Research Center, Liege University, Liege, Belgium
TABLE I
BindingAffinityValues(Ki,nM)of VariousSerotoninergic
Antagonistsfor DifferentNeurotransmitterReceptorSites (23) S2 D2 cri [@H]KetanserlnrHIHaIOperIdOI[@HJWB-
Altanserin0.13624.55Ketansenn0.6324011Setoperone0.372513Ritanserin0.
jected (fernoralvein) under light ether anesthesiawith S2antag
onists such as ketanserin (2.5 rng/kg), ritanserin (2.5 mg/kg),
pipamperone (10 mg/kg), and methysergide (2.5 mg/kg) or D antagonistssuch as sulpride(40 mg/kg), halopernide(20 mg/kg), and bromolisuride (0.4 mg/kg). Spiperone, a D2, S2 antagonist, was also used at 2 mg/kg, The drugs were dissolved in saline containing 5% alcohol and one equivalent of tartaric acid to
increasedrug solubility.The animalswerepretreated 1 hr before
injection of tracer and killed by decapitation 2 hr post-tracer
injection.
Studies on the Effect of Carrier To check the saturabiity of altanserin uptake in the frontal cortex, a region rich in S2 receptors sites, the frontal cortex-to cerebellum ratios were determined under light anesthesia in rats after intravenous injection (fernoralvein) of 100 @iCi[‘8F]altan serin at different specific activities (2 to 1000 mCi/@mol). The rats (2 for each specific activity) were killed by decapitation 2 hr
after injectionofthe radiotracer.
ChemicalFormof 1F in Rat Plasma
Four rats (200 g) were intravenously injected (fernoral vein) with 300 @Ciof [‘8F]altanserinunder light ether anesthesia (spe cific activity: 0.8 Ci/@imol).The blood (±5ml) was collected in heparmnatedtubes from the heart 4 hr after radiopharmaceutical
injectionand centrifugedfor 5 mm at 4000rpm. The plasmawas
diluted 12-fold with water at pH 4 (0.05 M HOAc) and slowly passed through a C-i 8 Sep Pak cartridge. After washing the
support with0.1%triethylaminein water(10 ml), the radioactiv
ity was eluted with a tetrahydrofuran and methanol mixture (25/ 75, 1.5 ml). The radioactive solution was then analyzed by HPLC
using a LichrosorbRP Select-Bcolumn (250 x 0.4 cm) eluted
with [CH3OH/THF/H2O (pH 4): 13/32.5/54.5] at a flow rate of 0.7 rnl/min. The extraction efficiency of the radioactivity was 95%—97%.
ChemicalFormof 18Fin Rat Brain
Rats (200 g, n=4) were intravenously injected (femoral vein) with 300 @Ciof [‘8F]altanserinunder light ether anesthesia. The animals were killed by decapitation 4 hr after injection of the radiotracer. Each brain was removed and homogenized using a
Polytrontissue disrupter with 4 ml of cold methanol (25). The
homogenate was centrifuged for 4 mm and the supernatant decanted. The pellet was again homogenized with 4 ml of cold methanol and centrifuged. A small amount of unlabeled altan
serinwasadded to the combined methalonicextracts(extraction
efficiency > 90%) and an aliquot of this solution analyzed in
HPLCasdescribedforplasmaanalysis.
RESULTS
Radiosynthesis
The radiolabeing of [‘8F]altanserinwas carried out by
nucleophilic substitution of [‘8F]fluorideon 3-(2-(4-(4-
thtrobe@oyl@l-piperidinyl)-propyl)-l,2-dihydro-2-thioxo
4-(3H)quinazolinone (mtroaltanserin (F), Fig, 1). This pre cursor was synthesized from p-nitrobenzoyl-4-piperidine
(A) according to the route shown in Figure 2. The prepa
ration of this substrate(A) involved a multi step synthesis
starting with isonipecotic acid as previously described (16).
This cold substrate (A) was coupled with @3-chloroethyl
ethylcarbamate followed by hydrolysis and cyclization of
nitroaltanserin, previously synthesized from p-mtrobenzoyl-4-
piperidine.
Biodistributlon Study
The in vivo biodistributionwas measuredin femaleWistar
rats (180—250g)intravenouslyinjected(femoralvein)under light
ether anesthesia with 80-150 iCi of['8F]altanserin (specific acti vity: 0.6-1.2 Ci/smol). Rats were killed at various times (5 mm, 1 hr, and 4 hr after injection) by cardiac excision under ether
anesthesia.The organs were removed and counted with a GeLi
detector connected with a multi channel analyzer. The percent ages of injected dose per gram of organ were calculated by comparisonwith a referencesolution consistingof diluted sam pies ofthe injected compound.
Brain Biodlstrlbution Study
The rat brainbiodistributionwas determinedby dissecting,
weighing,and counting samples (automatic gamma sample chan
ger, BertholdBF 5300) from differentbrain regions(frontal
cortex, striatum, thalamus, cerebellum) after femoral injection of
50—100@Ciof['8F]altanserin(specificactivity:0.6—1.2Ci/@mol).
FemaleWistarrats(180—250g) wereinjectedunderlightether
anesthesia and killed by decapitation. The results are expressed aspercentageof injecteddosepergram(%ID/g) of tissue.From
thesedata, regions-to-cerebellumratioswerecalculatedand corn
BlockingExperiments
Theselectivityof['8F]altanserinbindingto serotoninS2recep
tor sites was examined as follows.Rats were intravenouslyin
FIGURE1. Radiolabelingof [18F]aitansennStartingfromits
nitroprecursor.
0
(K/222J1F DMSO, MIcro Way., 5@
Altansenn for the Study of Serotonin Receptors with PET •Lemaire et al 2267
H
@@ @.##‘ @F C cH
II
0
H
—
]@LFrornamcortSx/cSrebmmum
injection). The activity in the cerebellum was low and also
relatively stable from 1 to 4 hr after injection, 0. 12% ±
0.03% and 0.09% ±0.0 1% ID/g of tissue, respectively.
The time course of tissue-to-cerebellumratios is shown
in Figure 5. The ratios of striatum and thalamus to cere
bellum were low (3.6 ±0.4 and 1.8 ±0.2, 2 hr after
injection, respectively). By contrast, the frontal cortex-to
cerebellum ratio increased strongly with time and reached
a plateau of 10.8 ±1.3 at 2 hr postinjection.
The specificity ofthe in vivo [‘8F]altanserinbinding was
evaluated by blocking experiments using S2 and D2 antag
onists. The results are summarized in Table 4. Drugs with
high affinity for serotonin S2 receptor sites (ketanserin,
pipamperone,ritanserin)(23,29) and spiperone, which has
a mixed D2—S2affinity (23), strongly blocked the [‘8F
altanserin accumulation in the frontal cortex and striatum.
Methysergide, which is also very specific for S@sites (23),
partially prevented [‘8F]altanserinuptake in these two brain
regions. Drugs showing higher affinity for D@than for S@
receptorsitessuchashalopemide(30,31)and bromolisuride (32) weakly decreased frontal cortex and striatum-to-cere
bellum ratios. Sulpiride, a specific D2 antagonist (31), did
not modify significantlythe FC/CB and the ST/CB ratios.
The frontal cortex-to-cerebellum ratio was drastically
influenced by the specific activity of the radiolabeled com
pound and decreasedwith the addition ofearrier. Ratios of
12.2 and 3.1 were found for [‘8F]altanserininjection with
specific activities of 1 Ci/@mol and 2 mCi/@mol respec
tively. The in vivo stability of['8F]altanserin was determined by
studying the percentage of unchanged radiopharmaceutical
in ratbrainand blood 4 hrafterradiotracerinjection. Under
the conditions described in the Materials and MethOdS
section, the HPLC analysis showed in both cases a main
radioactive peak corresponding to [‘8F]altanserin.This peak
contained more than 85% and 96% ofthe extractedradio
activity from blood and brain samples@respectively.
DISCUSSION
Synthesis
As shown in Table 2, the radiofluorination reaction with
bromoaltanserin as precursor gave a very poor radiochem
ical yield of [‘8F]altanserin,predicting nevertheless the pos
sibility of a higher fluorination yield by direct nucleophilic
‘8F-N02displacement (33). The critical prerequisiteof this
work was therefore the preparation of the nitroaltanserin
precursor (F), which has not been previously reported.
The radiochemicalyields obtained with the nitro deriva
tive were compared using conventional and microwave
heatingconditions. Although the automation of microwave
heating is technically complicated, this technique led to a
reduced reaction time and higher reproducible radiochem
ical yields. Furthermore, the use of lower starting amounts
of precursorled to easier purificationsteps.
Optimization of the HPLC conditions was achieved on
a LichrosorbRP Select-B Merck column using the method
‘. -.--^ —@0-@—Fron@@Coflx —@0——SMs@@m @ —•-
1.
30 SO SO (^120) ISO 150 210 240 limo (mm)
FIGURE4. Timecourseof‘8Fradioactivityinratfrontalcortex,
stnatum,thalamusandcerebellumafterintravenousinjectionof [18F]altanserin.Data are mean%dose/g ±s.d. (n=6).
percent ofthe administrated dose localized rapidly into the
brain and remained constant for more than 1 hr indicating
a significant retention by brain tissue. O@@ce of radio
activity took place at 4 hr after injection for all tissues. Five
minutes after injection, the %ID/g ofbone was 0.18%. This
value increased slowly with time to reach 0.31% at 4 hr
postinjection. Platelets which are known to contain S recep
tors (27) were isOlated from 1 ml of plasma as described
previously by Boyum (28). Values of 1.3% and 2% of the
percentage of the ID/g of blood were found in platelets at
5 mm and 4 hr after injection respectively.
The time course of ‘8Fradioactivityin cerebellum (CB),
frontal cortex (FC), striatum (ST), and thalamus (U) was
determined in rats (n=6). As shown in Figure 4, the radio
activity in the FC decreased slowly with time from 1.14%
±0.18% ID/g of tissue at 5 mm to a value of 1.00% ±
0.1 1%at 60 mm and remained relatively constant there
after. The striatum and thalamus displayed lower uptakes
(0.39 ±0.05 and 0.18 ±0.03, respectively, at 1 hr after
p
I
FIGURE5. Timecourseof tissue-to-cerebellumratiosof 18F
radioactivity after Intravenousinjection of [18F]aItansermn.Data are meanratios±s.d.(n=6).
14
@^ • 12
U^ • @ (^) 3 10
2-
J S
S
4 Strlatum/C.r.b.Imum mSmsmuslcsr.bsmmum^ j. 2
0 30 50 50 120 150 iSO 210 240 TmmSSft.t mnl.ctmon (mmn)
Altanserin for the Study of Serotonin Receptors with PET •Lemaire et al (^2269)
ColdligandK,
(nM)
32D2Injected
dose (mg/kg)Frontal
cortex! CerebellumReferencesNo cerebellumStriatum/
0.2n.c.a.competingligand100 @Ci10.3 ±0.63.4 ± Ci/Mmol)Ketansermn0.632402.51.6±0.21.5±0.223,29Ritansermn0.28222.51[18F]altansenn(0.
0.223Pipamperone0.9496101.4±0.21.5±0.223,29Methysergide1 .5 ±0.21 .4 ±
29Spiperone0.640.2621 .301402.53.2 ±0.52.0 ±0.323, 29Sulpiride>100031409.8 .5 ±0.21 .3 ±0.223, 0.331HalOpemide2203.1207.1 ±0.73.3 ± 31Bromolisuride—0.30.46.4 ±0.72.9 ±0.230, 0.2322Kd.The ±0.42.8 ±*
ratswerepretreated1hr beforeinjectionof tracerandkilled2 hr post [18F]altansenninjection(n= 4).
TABLE 4
Effectof DifferentCompetingS2and D2ReceptorLigandson the RegionalDistributionof [18F]Altansenn
previously reported(34,35). The pH was ofcritical influence
on the retention time of['8F]altanserinand its nitroprecur
sor. On the analyticalscale, a good separationbetween the
two derivatives was obtained in less than 15 mm using a
solvent mixture CH3OH/THFIH2O at pH 4 (12.6/32.4/
55). Unfortunately on the preparative scale, due to the
appearance of side products in the labeling step, a satisfac
tory purificationof['8F]altanserinwas only achieved at pH
5, which increased the retention times of the labeled and
unlabeled compounds (Fig. 3).
Animal Experiments
In order to validate the use of [‘8F]altanserinas a sero
toninergic radioligand for in vivo binding assays, animal
experimentswere carriedout.
Significant uptake of [‘8F]altanserinwas observed in the
frontal cortex, a brain region known to contain high S@(36)
and low a, receptordensities. A slight but significantaccu
mulation of['8F]altanserinwas found in the striatum,which
contains many more 1)2 than S2 sites. The [‘8F]altanserin
uptake in the thalamus displaying a, receptordensity, was
very low.
The critical point of this study was to differentiatethe
binding of [‘8F]altanserinto S2 from a, sites in the frontal
cortex and the binding to S@from 1)2sites in the striatum.
Regarding the possible binding of['8F]altanserin to a, recep
tors, the only known pure a, antagonist prazosin, widely
used in vitro, has a poor penetration into the brain (Leysen
JE,personalcommunication).This compound was not used
in our experiments. However, as shown in Table 1, altan
serin, like ketanserin, has a much more lower binding
affinity for a1 than for S2receptor sites.
Regarding the differentiation between [‘8F]altanserin
binding on S2and 1)2sites, several serotomn S@and dopa
mine D2 receptorblockerswere chosen with respectto their
in vitro biochemical properties(Ks,K@)shown in Table 4.
The doses of pretreatmentwere selected accordingto pub
lished data [Table 4, (29,32)]. When this information was
not available, the injected doses were sufficient to induce
catalepsy in rats (halopemide).
Theoretically, [‘8F]altanserinbinding on S@receptors sites
in the frontal cortex and in the striatum should be com
pletelyblockedby pretreatmentwith specificS@antagonists.
Our data clearly demonstrated that all the S@antagonists
and particularly ketanserin (Ks: nM for S@and D@receptor
sites: 0.63-240) strongly prevented [‘8F]altanserinaccumu
lation both in the frontal cortex and in the striatum, sug
gestingan [‘8F]altanserinbinding to S@receptor sites in those brain regions. Furthermore, [‘8F]altanserinuptake in
the striatum seems to occur on S@receptor sites since
ketanserin, as demonstrated by Suehiro et al. (37) and
Maziere Ct al. (32), did not modify in this region of the
brain selective binding of specific D2 ligands to dopamine
D2 receptor sites (N-[―C]methyl-benperidol and [76Br]
bromolisuride). Spiperone, which has a mixed D2—S@affin
ity, showed the same behavior. The incomplete blockage measured after pretreatment with methysergide could be
explained by the lack ofsaturation ofthe S@sites, even with
an injection ofthe ligand at a dose of 10 mg/kg.
In principle, the [‘8F]altanserinbinding on S@receptor
sites in the frontal cortex and striatum should not be
influenced by pretreatment with specific 1)2 antagonists.
The biochemical pattern of halopemide and bromolisuride (30,31,32) shows that these drugs exhibit more affinity for dopamine D2 than serotomn S@receptors. However, a par
tial occupancy of S2 sites may be expected when these
ligands are used at doses required to reach saturation of D@
receptorsites(i.e., 30 mg/kg and 0.4 mg/kg. respectively).
Our results displayed this feature, but pretreatment with
halopemide and bromolisuride did not influence more sig
nificantly the ST/CB than the FC/CB ratio suggesting also
a selective [‘8F]altanserinbinding in the striatumon S@sites.
Sulpiride is highly specific but has a rather low dopamine
D2 receptor binding affinity (31). Its penetration into the
brain is very poor (36) and this drug caused the same
problems in our pretreatmentinvestigationsas those men
@ 2270 The Journalof NuclearMedicine•Vol.32 •No. 12 December
the serotonergic5-HT2‘@PtO@Sin the human cerebralcortex using positron emission tomography and ‘@F-1abe1edSetoperone. J Neurochem 1990;54:1744—1754.
18. Wong DF, Lever JR. Harug PR, et a). Localization of serotonin 5-HT receptors in living human brain by positron emission tomography using N1([@C]inethy1)-2.Br.I.SD. Synapse 1987;1:393—398. 19. LeveriR, Dannals BY, Wilson AA, et aLSynthesis and in vivo characteriza tion of D(+XN1-―Cmethyl)-2-Br-LSDa radioligandfor positronemission tomographic studies of serotonin 5-HT2 receptoi@. Nuc/ Med Biol 1989;16:697—704. 20. LeysenJE, Gommeren W. Drug-receptordissociationtime, new tool for drug research: receptor binding affinity and drug-receptor dissociation profiles of serotonin-S2, dopamine D@,histamine H,, antagonists, and opiates. Dn@gDev Res 1986;8:119—131.
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Specific Activfty
The specificactivityof [‘8Fjaltanserinwas determined on an
analyticalreverse-phaseHPLC column (LichrosorbRP Select-B
column (250 x 4.6 mm). The mobile phase was CH3OHIFHF/
H2OpH 4 (12.6/32.4/55)witha flowrateof 0.8 ml/min.Under
these experimental conditions, the retention time of['8F]altanserin
was 10mm. The UV detectorwitha 10-mmanalyticalcellwasset
at 254 nm and the area of the UV absorbance peak of [‘@F] altanserin was determined by an automated integrator (Shimadzu
C-R5AChromatopac).A calibrationcurvewasdeterminedwith
authenticreferencesamples.
ACKNOWLEDGMENTS
The authorswish to thank Dr. M. Janssenofianssen Pharma ceutica, Beerse, Belgium, for graciously providing samples of altan
serin,bromoaltanserin,and nitrobenzoylpiperidine.They are also
gratefulto Dr. J. E. Leysen(JanssenPharmaceutica)for providing
methysergide,pipamperone,halopemide,and ketanserinand also to Dr. B. Mazière(Orsay)forthe giftofbromolisuride.The authors also thank Dr. D. Comar, Dr. B. Sadzot, and Dr. E. Salmon for
their helpfuldiscussions.
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(^2272) The Journalof NuclearMedicine•Vol.32 •No. 12 •December
